Position effect of the excision frequency of the Antirrhinum transposon Tam3: implications for the degree of position-dependent methylation in the ends of the element.

We identified eight independent Tam3 copies residing in the same Antirrhinum majus genome. All the copies showed excision at 15 degrees C, but not at 25 degrees C. Under conditions promoting excision, each copy appeared to transpose in the leaves and flower lobes with a nearly constant frequency, whereas individual transposition abilities varied widely: the most active copy had an excision frequency more than 100-fold greater than that of the least active one. Despite the different transposition abilities, the structures of the eight Tam3 copies were almost identical. These results made it clear that the transpositional ability of Tam3 is regulated by chromosomal position, but they do not imply position-dependent transposase activity. The position effect of the Tam3 transposition was found to be correlated to the methylation state of the copy's end regions: DNA methylation in the Tam3 end regions tended to suppress the excision activity, and the degree of methylation was dependent on the chromosomal position. Our results also provide evidence of de novo methylation provoked by transposition of the endogenous element. We propose a mechanism of transpositional regulation of plant transposons that responds to the degree of methylation as determined by chromosomal position.

Earlier detection of human immunodeficiency virus type 1 p24 antigen and immunoglobulin G and M antibodies to p17 antigen in seroconversion serum panels by immune complex transfer enzyme immunoassays.

For earlier diagnosis of human immunodeficiency virus type 1 (HIV-1) infection, the sensitivities of immune complex transfer enzyme immunoassays for HIV-1 p24 antigen and antibody immunoglobulin G (IgG) to HIV-1 p17 antigen were improved approximately 25- and 90-fold, respectively, over those of the previous immunoassays by performing solid-phase immunoreactions with shaking and increasing the serum sample volumes, and immune complex transfer enzyme immunoassay of antibody IgM to p17 antigen was also performed in the same way as the improved immunoassay of antibody IgG to p17 antigen. By the improved immunoassays, p24 antigen and antibody IgG to p17 antigen were detected earlier in 32 and 53%, respectively, of the HIV-1 seroconversion serum panels tested than before the improvements, and p24 antigen was detected as early as or earlier than HIV-1 RNA by reverse transcriptase-PCR (RT-PCR) in all of the panels tested. In 4 panels out of 19 tested, antibody IgG to p17 antigen or both antibodies IgG and IgM to p17 antigen were detected earlier than p24 antigen and RNA, although the antibody levels declined slightly before their steep increases usually observed after p24 antigen and RNA. Thus, the window period in diagnosis of HIV-1 infection can be shortened by detection of p24 antigen with the improved immunoassay as much as by detection of RNA with RT-PCR and, in some cases, more by detection of antibodies IgG and IgM to p17 antigen with the improved immunoassays than by detections of p24 antigen with the improved immunoassay and RNA with RT-PCR.

Recombinant p51 as antigen in an immune complex transfer enzyme immunoassay of immunoglobulin G antibody to human immunodeficiency virus type 1.

An ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) of antibody immunoglobulin G (IgG) to human immunodeficiency virus type 1 (HIV-1) has been developed using recombinant HIV-1 reverse transcriptase (rRT) as antigen. However, some disadvantages were noted in the use of rRT as antigen: rRT was produced only with low efficiency in widely used strains of Escherichia coli using a rather long DNA fragment (3,012 bp) of the whole HIV-1 pol gene, and it was impossible to produce fusion proteins of RT for simple purification, since rRT is a heterodimer of p66 and p51. In this study, recombinant HIV-1 p51 and p66 with Ser-Ser at the N termini (Ser-Ser-rp51 and Ser-Ser-rp66) were produced in E. coli as fusion proteins with maltose binding protein containing a factor Xa site between the two proteins and were purified after digestion with factor Xa. Ser-Ser-rp51 was produced in larger amounts and purified in higher yields with less polymerization than Ser-Ser-rp66. Polymerized Ser-Ser-rp66 tended to be precipitated on mercaptoacetylation for conjugation to beta-D-galactosidase (used as a label) and showed higher nonspecific and lower specific signals in an immune complex transfer enzyme immunoassay of antibody IgG to HIV-1 than Ser-Ser-rp51. The signals for serum samples of HIV-1-seropositive subjects by immune complex transfer enzyme immunoassay of antibody IgG to HIV-1 using Ser-Ser-rp51 as antigen (Y) were well correlated to those obtained using rRT as antigen (X) (log Y = 0.99 log X + 0.23; r = 0.99). Thus, the use of rp51 as antigen was advantageous over that of rp66 and rRT in an immune complex transfer enzyme immunoassay of antibody IgG to HIV-1.

Development of highly sensitive enzyme-linked immunosorbent assays for hepatocyte growth factor/scatter factor (HGF/SF): determination of HGF/SF in serum and urine from normal human subjects.

A sandwich enzyme-linked immunosorbent assay (ELISA) using rabbit anti-hepatocyte growth factor (HGF) IgG for human HGF, also known as the scatter factor, has previously been developed for determining increases in serum HGF levels in various liver diseases. The sensitivity limit of the ELISA is, however, approximately 0.2 ng/ml sample, and HGF concentrations in about 50% of normal subjects are not accurately measurable by this method, because the mean level of HGF in normal serum is close to the sensitivity limit. In the present study, chicken Fab' from egg yolk anti-HGF immunoglobulin Y and rabbit Fab' from rabbit anti-HGF IgG were conjugated with beta-D-galactosidase. With these conjugates as the second antibodies, we developed two sandwich ELISAs for human HGF and found that the sensitivities were about 20 pg/ml with the former conjugate and 2 pg/ml with the latter. The HGF concentration in sera from 138 normal subjects determined by the ELISA with the rabbit conjugate was 244+/-65 (SD) pg/ml serum, and it correlated very well with the number of leukocytes. Moreover, the ELISA with the rabbit conjugate permitted the determination of HGF levels in urine from normal subjects without first concentrating the sample. The determination of HGF in various biological fluids other than blood and urine by these ELISAs may aid the diagnosis and prognosis of various diseases.

Preparations of recombinant HIV-1 p66 antigen to improve the specificity of immune complex transfer enzyme immunoassay of antibody IgG to HIV-1 reverse transcriptase.

Recombinant HIV-1 p66 (rp66, a subunit of reverse transcriptase (RT), a heterodimer of p66 and p51) was produced in Escherichia coli in three different ways. First, rp66 was produced as a part of the fusion protein of lacZ protein and HIV-1 pol protein consisting of three components: protease (p10), RT (p51/p66), and integrase (p31), and was released from the fusion protein by the protease (pol-rp66). Second, rp66 with Ser-Ser at the N-terminus was produced as a fusion protein with maltose-binding protein containing a factor Xa site between the two proteins (MBP-Ser-Ser-rp66) and was released from the fusion protein by factor Xa (Ser-Ser-rp66). Third, rp66 with Met-Gly at the N-terminus was produced in transformed cells (Met-Gly-rp66). The recombinant proteins were purified from sonic extracts of transformed cells by ammonium sulfate fractionation and various column chromatographies. MBP-Ser-Ser-rp66 and Met-Gly-rp66 were readily purified in sufficient amounts for labeling with 2, 4-dinitrophenyl groups and beta-D-galactosidase from E. coli, but pol-rp66 and Ser-Ser-rp66 were not for enzyme-labeling. Ser-Ser-rp66 was not only polymerized but also degraded to considerable extents. The purified preparations were labeled with 2,4-dinitrophenyl groups and beta-D-galactosidase and were tested in immune complex transfer enzyme immunoassay of antibody IgG to HIV-1 RT using serum samples from 600 HIV-1 seronegative and 30 HIV-1 seropositive subjects. Among various combined uses of the two labeled preparations, the uses of 2,4-dinitrophenylated MBP-Ser-Ser-rp66 and pol-rp66 with beta-D-galactosidase-labeled Met-Gly-rp66 showed the highest (99.8%) and the second highest (99.5%) specificities, which were higher than that with the labeled preparations used in the previous study (98. 0%).

Ultrasensitive immune complex transfer enzyme immunoassay of HIV-1 p24 antigen with less serum interference using 2,4-dinitrophenyl-anti-HIV-1 p24 IgG and indirectly immobilized (anti-2,4-dinitrophenyl group) Fab..

In the immune complex transfer enzyme immunoassay previously reported, the immune complex consisting of 2,4-dinitrophenyl-biotinyl-bovine serum albumin-affinity-purified rabbit anti-HIV-1 p24 Fab' conjugate, HIV-1 p24 antigen and monoclonal mouse anti-HIV-1 p24 Fab'-beta-D-galactosidase conjugate was trapped on polystyrene beads coated directly with affinity-purified (anti-2,4-dinitrophenyl group) IgG and was transferred to polystyrene beads coated with biotinyl-bovine serum albumin and streptavidin. The serum volume used was limited to 10 microL due to serious serum interference, and the detection limit of HIV-1 p24 antigen was 240 fg/mL serum. In the present study, HIV-1 p24 antigen was incubated simultaneously with 2,4-dinitrophenyl-affinity-purified rabbit anti-HIV-1 p24 IgG and monoclonal mouse anti-HIV-1 p24 Fab'-beta-D-galactosidase conjugate in the presence of excess nonspecific rabbit IgG. The immune complex of the three components formed was trapped on polystyrene beads coated successively with biotinyl-bovine serum albumin, streptavidin and biotinyl-affinity-purified (anti-2,4-dinitrophenyl group) Fab'. After washing, the immune complex was eluted from the polystyrene beads with excess epsilonN-2,4-dinitrophenyl-L-lysine and transferred to polystyrene beads coated with affinity-purified goat (antirabbit IgG) IgG. The serum volume used was increased to 90 microL with only slight serum interference, and the detection limit of HIV-1 p24 antigen was lowered 9-fold to 26 fg/mL serum.

Use of indirectly immobilized recombinant p17 antigen for detection of antibodies to HIV-1 by enzyme immunoassay.

Recombinant HIV-1 p17 antigen (rp17) and maltose binding protein-rp17 fusion protein (MBP-rp17) were immobilized in different ways: rp17 and MBP-rp17 were immobilized directly onto polystyrene beads by physical adsorption, and biotinyl-rp17, biotinyl-MBP-rp17, and 2,4-dinitrophenyl (DNP)-MBP-rp17 were immobilized indirectly onto polystyrene beads, which had been coated with streptavidin alone, with biotinyl-bovine serum albumin and streptavidin and with (anti-2,4-dinitrophenyl group) IgG. These immobilized antigens were tested by incubation with diluted serum from an HIV-1 seropositive subject in the absence and presence of serum from HIV-1 seronegative subjects and, after washing, with rp17 beta-D-galactosidase conjugate. Higher positive signals (fluorescence intensities for bound -beta-D-galactosidase activity) and less serum interference were obtained with indirectly immobilized antigens than with directly immobilized ones. Enzyme immunoassay using biotinyl-MBP-rp17 indirectly immobilized onto polystyrene beads, which had been coated sequentially with biotinyl-bovine serum albumin and streptavidin, was approximately 1,000-fold more sensitive than that using directly immobilized rp17 antigen and Western blotting for p17 band. This enzyme immunoassay indicated positivity in HIV-1 seroconversion serum panels as early as or even earlier than conventional methods and considerably earlier than Western blotting for HIV-1 p17 band. In addition, the sensitivity was further improved approximately 10-fold by incubation with shaking for immunoreactions and by increase of both the number of polystyrene beads and the volume of serum samples used per assay.

Drosophila tRNAs hyperprocessed in vitro by ribonuclease P.

In transposon copia-related retrovirus-like particles of Drosophila, a 5' half fragment produced by the cleavage of mature initiator methionine tRNA is used as the primer for minus-strand reverse transcription. This cleavage is called hyperprocessing. We have previously reported that the catalytic RNA subunit of RNase P catalyzes this hyperprocessing in vitro and that this cleavage is dependent on the occurrence of an altered conformation of the tRNA substrate. Here, we found that other mature tRNAs of Drosophila were also hyperprocessed by M1 RNA in vitro and that some of such tRNAs were probably alanine and histidine tRNAs. Here we report these two tRNAs can also adopt their alternative conformations very similar to that of initiator methionine tRNA.

Immune complex transfer enzyme immunoassay for antibody IgM to HIV-1 p17 antigen.

The immune complex transfer enzyme immunoassay for antibody IgM to HIV-1 p17 antigen is described. Serum samples containing antibody IgM to HIV-1 p17 antigen were incubated simultaneously with 2,4-dinitrophenyl-bovine serum albumin-recombinant p17 (rp17) conjugate and rp17-beta-D-galactosidase conjugate, and the immune complex formed comprising the three components was trapped onto colored polystyrene beads coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG. Subsequently, the immune complex was transferred to white polystyrene beads coated with monoclonal mouse (antihuman IgM) IgG in the presence of excess of epsilonN-2,4-dinitrophenyl-L-lysine. The signal for antibody IgM to p17 antigen was the fluorescence intensity by fluorometric assay of beta-D-galactosidase activity bound to the white polystyrene beads. The periods of time required for the formation, trapping, and transferring of the immune complex comprising the three components were more than 4 hr, 2 hr, and 3 hr, respectively. The immunoassay developed was shown to be specific by inhibition of transferring the immune complex in the presence of excess of nonspecific IgM but not IgG. Signals for antibody IgM to p17 antigen with serum samples of HIV-1 seroconversion serum panels,--that is, with serum samples in early stages of the infection--tended to be higher than those with serum samples from HIV-1 asymptomatic carriers probably long after the infection and patients with ARC and AIDS. In contrast, signals for antibody IgG to p17 antigen with serum samples of HIV-1 seroconversion serum panels tended to be higher than signals for antibody IgM to p17 antigen but were much lower than signals for antibody IgG to p17 antigen with serum samples from HIV-1 asymptomatic carriers and patients with ARC and AIDS.

Sensitive enzyme immunoassay of antibodies to HIV-1 p17 antigen using indirectly immobilized recombinant p17 for diagnosis of HIV-1 infection.

Recombinant p17 (rp17) antigen of HIV-1 and maltose binding protein-rp17 fusion protein (MBP-rp17) were immobilized onto polystyrene beads in different ways: rp17 and MBP-rp17 were immobilized directly onto polystyrene beads by physical adsorption; biotinyl-rp17 and biotinyl-MBP-rp17 were immobilized indirectly onto streptavidin-coated polystyrene beads; and 2,4-dinitrophenyl (DNP)-MBP-rp17 was immobilized indirectly onto (anti-DNP) IgG-coated polystyrene beads. These directly and indirectly immobilized antigens were incubated with urine samples containing antibody IgG to p17 antigen and subsequently with rp17-beta-D-galactosidase conjugate or (anti-human IgG gamma-chain) Fab'-beta-D-galactosidase conjugate. Beta-D-galactosidase activity bound to the polystyrene beads was assayed by fluorometry. When rp17-beta-D-galactosidase conjugate was used, signals (fluorescence intensities for bound beta-D-galactosidase activity) were much higher with the indirectly immobilized antigens than those with the directly immobilized antigens. By experiments using (anti-human IgG gamma-chain)Fab'-beta-D-galactosidase conjugate, the binding of rp17-beta-D-galactosidase conjugate to antibodies against p17 antigen bound to directly immobilized rp17 antigen was shown to be seriously limited as compared with that to antibodies against p17 antigen bound to indirectly immobilized DNP-MBP-rp17. When rp17-beta-D-galactosidase conjugate and serum samples were used, serum interference was much less with indirectly immobilized DNP-MBP-rp17 than with directly immobilized rp17 antigen, and the sensitivity of enzyme immunoassay for antibody IgG to p17 antigen using indirectly immobilized DNP-MBP-rp17 was 1,000- to 3,000-fold higher than that of enzyme immunoassay using directly immobilized rp17 antigen and Western blotting for p17 band. This sensitive enzyme immunoassay indicated positivity in HIV-1 seroconversion serum panels as early as conventional methods for antibodies to HIV-1 and earlier than Western blotting for p17 band.

Sensitive enzyme immunoassay for anti-beta-lactoglobulin IgG in serum.

A sensitive enzyme immunoassay for anti-beta-lactoglobulin immunoglobulin G (IgG) in serum is described. Serum containing anti-beta-lactoglobulin IgG was reacted simultaneously with 2,4-dinitrophenyl-bovine serum albumin-beta-lactoglobulin conjugate and beta-lactoglobulin-peroxidase conjugate. The complex formed from the three components was trapped onto polystyrene balls coated with anti-2,4-dinitrophenyl group IgG, eluted with epsilon N-2,4-dinitrophenyl-L-lysine and transferred to polystyrene balls coated with anti-human IgG.gamma-chain IgG. Bound peroxidase activity was determined by fluorometry. This enzyme immunoassay was 100- to 1000-fold more sensitive and more reliable than the enzyme-linked immunosorbent assay (ELISA). Anti-beta-lactoglobulin IgG was detected in 91% of healthy subjects using this method.

Immune complex transfer enzyme immunoassay for antibody IgG to HIV-1 gp41 antigen using synthetic peptides as antigens.

The immune complex transfer enzyme immunoassay for antibody IgG to HIV-1 gp41 antigen was developed using two synthetic peptides. An aliquot (10 microl) of serum samples from HIV-1 seropositive subjects was incubated simultaneously with 2,4-dinitrophenyl-bovine serum albumin-synthetic HIV-1 gp41 peptide conjugates and synthetic HIV-1 gp41 peptide-beta-D-galactosidase conjugates and subsequently with colored polystyrene beads coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG to trap the immune complexes formed comprising the three components. After washing, the colored polystyrene beads were incubated with white polystyrene beads coated with affinity-purified (anti-human IgGgamma-chain) IgG in the presence of epsilonN-2,4-dinitrophenyl-L-lysine to transfer the immune complexes to the white polystyrene beads. Beta-D-Galactosidase activity bound to the white polystyrene beads was assayed by fluorometry. The formation, trapping and transferring of the immune complexes were completed within 0.5, 0.5 and 1.5 hr, respectively. Since each peptide appeared to react with its own specific antibody IgG, serum samples were tested by the equimolar combination of the two peptides. The lowest signals (fluorescence intensities for bound beta-D-galactosidase activity) for serum samples from HIV-1 asymptomatic carriers, patients with AIDS-related complex and patients with AIDS were 1490-, 2210- and 1460-fold, respectively, higher than the highest signal for serum samples from HIV-1 seronegative subjects. In five seroconversion serum panels, antibody IgG to HIV-1 gp41 antigen was detected as early as antibodies to HIV-1 detected by three currently commercially available methods.

Rapid and ultrasensitive enzyme immunoassay (thin aqueous layer immune complex transfer enzyme immunoassay) for HIV-1 p24 antigen.

The immune complex transfer enzyme immunoassay for HIV-1 p24 antigen was performed in three different ways (in the present immunoassays I, II, and III) within much shorter periods of time than previously reported. In the present (simultaneous) immunoassay I, p24 antigen was incubated simultaneously with 2,4-dinitrophenyl-biotinyl-bovine serum albumin-affinity-purified rabbit anti-p24 Fab' conjugate and monoclonal mouse anti-p24 Fab'-beta-D-galactosidase conjugate in a total volume of 19 microL for 15 min to form the immune complex comprising the three components. The reaction mixture was incubated with a polystyrene bead of 6.35 mm in diameter coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG for 5 min to trap the immune complex. After washing, the polystyrene bead was incubated with 35 microL of epsilonN-2,4-dinitrophenyl-L-lysine for 15 min to elute the immune complex (the first eluate) and, after removing the first eluate, with an additional 35 microL of epsilonN-2,4-dinitrophenyl-L-lysine for 1 min (the second eluate). The first and second eluates were incubated with a polystyrene test tube (12 x 75 mm) coated with streptavidin for 15 min. In the present (sequential) immunoassay II, a polystyrene bead of 6.35 mm in diameter successively coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG and 2,4-dinitrophenyl-biotinyl-bovine serum albumin-affinity-purified rabbit anti-p24 Fab' conjugate was incubated with p24 antigen in a total volume of 20 microL for 5 min and subsequently with monoclonal mouse anti-p24 Fab'-beta-D-galactosidase conjugate in a volume of 5 microL for 20 min. The immune complex formed on the polystyrene bead was transferred to a polystyrene test tube coated with streptavidin as described above. In the present (sequential) immunoassay III, p24 antigen was incubated with monoclonal mouse anti-p24 Fab'-beta-D-galactosidase conjugate in a total volume of 19 microL for 10 min and with a polystyrene bead of 6.35 mm in diameter coated successively with affinity-purified (anti-2,4-dinitrophenyl group) IgG and 2,4-dinitrophenyl-biotinyl-bovine serum albumin-affinity-purified rabbit anti-p24 Fab' conjugate for 20 min. The immune complex formed on the polystyrene bead was transferred as described above. The incubations were performed at room temperature either by shaking the polystyrene beads (one/assay) and the reaction mixtures in styrol test tubes (13.3 x 54 mm and 2.1 g) so as to randomly rotate the polystyrene beads or by rotating the polystyrene test tubes (12 x 75 mm) containing the reaction mixtures, so that small drops (19 to 70 microL) of the reaction mixtures evenly contacted all parts of the solid phase surfaces during the incubations (although they contacted only small parts of the solid phase surfaces at a time) to continuously mix thin aqueous layers covering the solid phase surfaces with the rest of the reaction mixtures. (Therefore, these immunoassays are called thin aqueous layer immunoassays.) The detection limits of p24 antigen by 1 hr assay of bound beta-D-galactosidase activity in the present immunoassays I, II, and III were 0.1, 0.2 and 0.1 amol/assay, respectively, and were slightly higher than or equal to that by the previously reported immune complex transfer enzyme immunoassay, in which the immune complex was formed for 4 hr, was trapped for 16 hr, and was transferred for 3 hr followed by 1-hr assay of bound beta-D-galactosidase activity. By 20-hr assay of bound beta-D-galactosidase activity, the detection limit of p24 antigen was further lowered to 10 zmol/assay in the present (simultaneous) immunoassay I and to 3 zmol/assay in the present (sequential) immunoassay III. However, the nonspecific reaction(s) with serum samples from HIV-1 seronegative subjects hampered the improvement of the detection limit by 20-hr assay of bound beta-D-galactosidase activity.

Rapid formation of the immune complexes on solid phase in the immune complex transfer enzyme immunoassays for HIV-1 p24 antigen and antibody IgGs to HIV-1.

In order to perform the immune complex transfer enzyme immunoassays for HIV-1 p24 antigen and antibody IgGs to HIV-1 p17, reverse transcriptase and gp41 antigens as rapidly as possible, methods for rapid formation of the immune complexes on solid phase are described. HIV-1 p24 antigen was reacted with monoclonal anti-p24 Fab'-beta-D-galactosidase conjugate at a high concentration and subsequently with polystyrene beads coated successively with affinity-purified (anti-2,4-dinitrophenyl group) IgG and 2,4-dinitrophenyl-biotinyl bovine serum albumin-affinity-purified anti-p24 Fab' conjugate. Antibody IgGs to HIV-1 were reacted with polystyrene beads coated successively with affinity-purified (anti-2,4-dinitrophenyl group) IgG and 2,4-dinitrophenyl-HIV-1 antigen conjugates and subsequently with HIV-1 antigen-beta-D-galactosidase conjugates. The periods of time used for the formation of the immune complexes comprising the three components on the polystyrene beads (15-30 min) were much shorter than those used in the previous immune complex transfer enzyme immunoassays (90-300 min), and the sensitivities of the present and previous immune complex transfer enzyme immunoassays were similar. The detection limit of the HIV-1 p24 antigen by the present and previous methods were similar (3 to 10 zmol/assay).

Potential of the immune complex transfer enzyme immunoassay for antigens and antibodies to improve the sensitivity and its limitations.

In order to reduce the nonspecific signal of noncompetitive solid phase immunoassays and to improve their sensitivities, the immune complex transfer enzyme immunoassay has been developed. Antigens to be measured were reacted with 2,4-dinitrophenyl-biotinyl-antibody Fab' and antibody Fab'-beta-D-galactosidase conjugate, and antibody IgGs to be measured were reacted with 2,4-dinitrophenyl-antigen and antigen-beta-D-galactosidase conjugate. The immune complexes formed comprising the three components were trapped onto colored polystyrene beads coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG. After washing, the immune complexes were eluted from the colored polystyrene beads with epsilonN-2,4-dinitrophenyl-L-lysine, and the eluates were incubated with white polystyrene beads coated with streptavidin for antigens and coated with affinity-purified (anti-human IgG gamma-chain) IgG for antibody IgGs to transfer the immune complexes. By this method, ultrasensitive enzyme immunoassays have been developed for HIV-1 p24 antigen and antibody IgGs to HIV-1 p17 and reverse transcriptase (RT). The nonspecific signals in the absence of the antigen and the antibody IgGs were reduced 300 to 15,000-fold by the immune complex transfer process, but the amounts of the immune complexes decreased only 1.8 to 3.1-fold by the immune complex transfer. As a result, the sensitivities for HIV-1 p24 antigen and antibody IgGs to HIV-1 p17 and RT were improved 100 to 5,600-fold by the immune complex transfer. The detection limit of HIV-1 p24 antigen by 20 hr assay of beta-D-galactosidase activity (10 zmol) was 4,000 to 17,000-fold lower than those obtained with currently available commercial kits. The improved sensitivities for antibody IgGs to p17 and RT by 20 hr assay of beta-D-galactosidase activity were 1 x 10(5) to 3 x 10(5)-fold higher than those of Western blotting for p17 and p66 bands. However, the nonspecific signals in the absence of antigens and antibody IgGs were enhanced to various degrees by two factors. In order to transfer the immune complexes more efficiently within shorter periods of time, the colored polystyrene beads were incubated with the white polystyrene beads in the presence of epsilonN-2,4-dinitrophenyl-L-lysine. Such direct contact between solid phases for trapping and transferring of the immune complexes significantly enhanced the nonspecific signals. In addition, the presence of human serum samples containing neither antigens to be measured nor antibody IgGs to be measured also enhanced the nonspecific signals to various extents. Namely, these two factors limited the effect of the immune complex transfer to improve the sensitivity by 20 hr assay of beta-D-galactosidase activity. By 1 hr assay of beta-D-galactosidase activity, the detection limit of HIV-1 p24 antigen using 10 microl of serum samples (0.24 pg/ml) was 40 to 80-fold lower than those obtained with currently available commercial kits using 100 to 200 microl of serum samples (10 to 20 pg/ml) and the detection limits of antibody IgGs to HIV-1 pl7 and RTwere 1 x 10(4) to 3 x 10(4)-fold lower than those by Western blotting for p17 and p66 bands. Finally, the immunoreactions involved in the immune complex transfer enzyme immunoassays--the formation, trapping, and transferring of the immune complexes--will be performed within 15 to 30 min.

Ultrasensitive and rapid enzyme immunoassay (thin aqueous layer immune complex transfer enzyme immunoassay) for antibody IgG to HIV-1 p17 antigen.

The immune complex transfer enzyme immunoassay for antibody IgG to HIV-1 p17 antigen was performed in two different ways (the present immunoassays I and II) within shorter periods of time than previously reported. In the present (simultaneous) immunoassay I, antibody IgG to HIV-1 p17 antigen in 10 microL of serum samples was incubated simultaneously with 2,4-dinitrophenyl-maltose binding protein-recombinant p17(rp17) fusion protein and rp17-beta-D-galactosidase conjugate in a total volume of 22 microL for 10 min to form the immune complex comprising the three components. The reaction mixture was incubated with a polystyrene bead of 6.35 mm in diameter coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG for 5 min in a styrol test tube (13.3 x 54 mm and 2.1 g) to trap the immune complex. After washing, the polystyrene bead was incubated with 30 microL of epsilonN-2,4-dinitrophenyl-L-lysine solution in a polystyrene tube (12 x 75 mm) coated with affinity-purified (antihuman IgG gamma-chain) IgG for 10 min to transfer the immune complex. In the present (sequential) immunoassay 11, a polystyrene bead of 6.35 mm in diameter coated successively with affinity-purified (anti-2,4-dinitrophenyl group) IgG and 2,4-dinitrophenyl-maltose binding protein-rp17 fusion protein was incubated in a styrol test tube (13.3 x 54 mm and 2.1 g) sequentially with antibody IgG to HIV-1 p17 antigen in 10 microL of serum samples in a total volume of 16 microL for 5 min and subsequently with rp17-beta-D-galactosidase conjugate in a volume of 10 microL for 5 and 10 min. The immune complex formed on the polystyrene bead was transferred to a polystyrene tube coated with affinity-purified (antihuman IgG gamma-chain) IgG for 5 and 10 min in the same way as in the present immunoassay I. During the incubations, the styrol test tubes containing the polystyrene beads and reaction mixtures were shaken, and the polystyrene test tubes were rotated with shaking, so that the polystyrene beads were rotated randomly, and small drops (16 to 30 microL) of the reaction mixtures evenly contacted all parts of the solid phase surfaces during the incubations, though only small parts of the solid phase surfaces were contacted at one time. The intent was to continuously mix thin aqueous layers of the reaction mixtures covering the solid phase surfaces with the rest of the reaction mixtures. (Therefore, these immunoassays were called thin aqueous layer immunoassays.) beta-D-Galactosidase activity bound to the polystyrene tubes was assayed by fluorometry for 30 and 60 min. The present immunoassays I and II, in which only 15 to 25 min were used for the immunoreactions, were as sensitive if not more so than the previous immune complex transfer enzyme immunoassay requiring 150 min for the immunoreactions. In these earlier immunoreactions, the immune complex comprising the three components formed by 30 min incubation was trapped onto two polystyrene beads (3.2 mm in diameter) coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG for 60 min, and was then transferred to two polystyrene beads (3.2 mm in diameter) coated with affinity-purified (antihuman IgG y-chain) IgG for 60 min in a total volume of 150 microL. Furthermore, the present (sequential) immunoassay 11 (and probably I) could become approximately 10 times more sensitive by assaying bound beta-D-galactosidase activity for a longer period of time (10 h), since beta-D-galactosidase activity, bound nonspecifically in the presence of serum samples from HIV-1 seronegative subjects, was considerably low.